The thermal dependence of the protein-sparing effect in rainbow trout (Oncorhynchus mykiss, Walbaum 1792).

The authors performed an instantaneous bioenergetic study with rainbow trout (Oncorhynchus mykiss) of 206.3 g ± 2.9 g in a group respirometer of nine 250 l tanks at five different water temperatures (12, 14, 16, 18, 20°C) to determine the optimal thermal condition for a maximal visualization of the protein-sparing effect. Twelve fish per tank were tested at a stocking density of 9.94 kg m-3 ± 0.14 kg m-3 and fed three low-protein/high-energy diets with constant crude protein content of c. 35% and three different energy contents (17.35, 18.76, 20.50 MJ kg-1 ) once daily at a ration of 1.3% body weight (n = 3). Energy levels were increased by adding gelatinized wheat starch as a carbohydrate source and fish oil, canola oil and palmitin as lipid sources. Three different dietary digestible protein/digestible energy ratios (DP/DE: 20.38, 19.08, 18.09 mg kJ-1 ) were achieved by replacing bentonite as a non-nutritive filler with carbohydrates and lipids. Oxygen consumption and ammonia excretion were assessed to obtain the potentially retainable energy (RE) and ammonia quotient (AQ) as benchmarks for potential growth and protein-sparing effect. The results showed the lowest relative metabolic combustion of protein at 16.9°C ± 0.1°C. The authors determined this temperature to set the optimal thermal condition for the induction of a maximum protein-sparing effect in juvenile rainbow trout. Increasing the DP/DE ratio significantly altered the magnitude of the relative metabolic protein use but had no effect on its interactions with temperature. The authors were able to reduce average metabolic fuel use of protein across diets from 16.2% ± 2.3% at 12°C to 8.0% ± 1.2% at 16°C. This study found no relevant significant differences of RE with the environmental temperature.

Dietary Supplementation with Chromium DL-Methionine Enhances Growth Performance of African Catfish (Clarias gariepinus).

Sustainable aqua feeds have become an urgent necessity for future-oriented aquaculture sector development, and especially mineral supply could be limited when diets are being prepared with low amounts of animal-based sources. Since knowledge about the efficiency of organic trace mineral supplementation in different species of fish is limited, the effects of chromium DL-methionine in African catfish nutrition were evaluated. Four commercially based diets with increasing chromium DL-methionine supplementation (0, 0.2, 0.4, and 0.6 mg Cr kg-1) in the form of Availa-Cr 1000 were fed to African catfish (Clarias gariepinus B., 1822) in quadruplicate groups for 84 days. Growth performance parameters (final body weight, feed conversion ratio, specific growth rate, daily feed intake, protein efficiency ratio, and protein retention efficiency), biometric indices (mortality, hepatosomatic index, spleen somatic index, and hematocrit), and mineral retention efficiency were assessed at the end of the feeding trial. The specific growth rate was significantly increased in fish-fed diets with 0.2 mg Cr kg-1 and 0.4 mg Cr kg-1 supplementation in comparison with control and based on the second-degree polynomial regression analysis; supplementation with 0.33 mg Cr kg-1 was optimal in commercially based diets for African catfish. Chromium retention efficiency was reduced with increasing supplementation levels; however, the chromium content of the whole body was comparable to literature. The results suggest that organic chromium supplementation is a viable and safe supplement for diets to increase the growth performance of African catfish.

Characterization of arthralgia induced by PD-1 antibody treatment in patients with metastasized cutaneous malignancies.

BACKGROUND: PD-1 antibodies (PD1ab) are increasingly used in metastatic melanoma and other malignancies. Arthralgia is an underestimated side effect of PD-1 antibody treatment with unknown cause. Our aim was to characterize PD1ab-induced arthralgia. PATIENTS AND METHODS: We retrospectively included patients with metastatic cutaneous malignancies treated with pembrolizumab or nivolumab ± ipilimumab at the National Center for Tumor Diseases (Heidelberg) between 01/2013 and 09/2016. Arthralgia was characterized by laboratory diagnostics, imaging, and if indicated, rheumatologic consultation. RESULTS: 26 of 195 patients (13.3%) developed arthralgia. The median onset of symptoms was 100 days (7-780 days). Most frequently, arthralgia involved large joints (shoulders, knees) in a predominantly symmetrical pattern. Only two patients were seropositive for rheumatoid factor and/or anti-citrullinated protein antibodies. Ten patients developed the clinical picture of arthritis, with seven of them showing synovitis in MRI or PET/CT. Five patients showed inflammation in joints pre-damaged by osteoarthritis. In 11 patients arthralgia could not be specified. The majority of patients was satisfactorily treated with non-steroidal anti-inflammatory drugs (NSAIDs), 23.1% required additional low-dose corticosteroids and only 7.6% of our patients received further immunosuppressive treatment. Patients with arthralgia showed a better treatment response and improved PFS and OS. CONCLUSION: Arthralgia is frequent during PD1ab treatment. The clinical picture varies between synovitis of predominantly large joints, progressive osteoarthritis and arthralgia without evident joint damage. Vast majority of cases can be satisfactorily managed by NSAID and/or low-dose corticosteroids.

Isolation and characterization of native probiotics for fish farming.

BACKGROUND: Innovations in fish nutrition act as drivers for the sustainable development of the rapidly expanding aquaculture sector. Probiotic dietary supplements are able to improve health and nutrition of livestock, but respective bacteria have mainly been isolated from terrestrial, warm-blooded hosts, limiting an efficient application in fish. Native probiotics adapted to the gastrointestinal tract of the respective fish species will establish within the original host more efficiently. RESULTS: Here, 248 autochthonous isolates were cultured from the digestive system of three temperate flatfish species. Upon 16S rRNA gene sequencing of 195 isolates, 89.7% (n = 175) Gram-negatives belonging to the Alpha- (1.0%), Beta- (4.1%) and Gammaproteobacteria (84.6%) were identified. Candidate probiotics were further characterized using in vitro assays addressing 1) inhibition of pathogens, 2) degradation of plant derived anti-nutrient (saponin) and 3) the content of essential fatty acids (FA) and their precursors. Twelve isolates revealed an inhibition towards the common fish pathogen Tenacibaculum maritimum, seven were able to metabolize saponin as sole carbon and energy source and two isolates 012 Psychrobacter sp. and 047 Paracoccus sp. revealed remarkably high contents of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Furthermore, a rapid and cost-effective method to coat feed pellets revealed high viability of the supplemented probiotics over 54 d of storage at 4°C. CONCLUSIONS: Here, a strategy for the isolation and characterization of native probiotic candidates is presented that can easily be adapted to other farmed fish species. The simple coating procedure assures viability of probiotics and can thus be applied for the evaluation of probiotic candidates in the future.

Fatty Acid Composition and Fatty Acid Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature.

To sustainably produce marine fish with a high lipid quality rich in omega-3 fatty acids, alternative sources of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are being identified. Moreover, the use of bioactive compounds that would stimulate the in vivo fatty acid synthesis, such as resveratrol (RV), would reduce the dependence on fish oil in aquafeeds. Gilthead sea bream (Sparus aurata) were fed four experimental diets combining two fish oil levels (6% dry matter (DM); 2% DM) with or without 0.15% DM resveratrol supplementation (F6, F2, F6 + RV, F2 + RV) for two months. Additionally, the fish were challenged either at 19 °C or 23 °C. A higher water temperature promoted their feed intake and growth, resulting in an increased crude lipid content irrespective of dietary treatment. The fatty acid composition of different tissues was significantly affected by the holding temperature and dietary fish oil level. The dietary RV significantly affected the hepatic EPA and DHA content of fish held at 19 °C. The observed effect of RV may be partly explained by alterations of the mRNA steady-state levels of ∆6-desaturase and ß-oxidation-related genes. Besides the relevant results concerning RV-mediated regulation of fatty acid synthesis in marine fish, further studies need to be conducted to clarify the potential value of RV to enhance fillet lipid quality.

The effect of diet, temperature and intermittent low oxygen on the metabolism of rainbow trout.

An automated respirometer system was used to measure VO2, protein catabolism as ammonia quotient and the energy budget to evaluate whether the crude protein content of a standard protein (SP) diet (42·5 %) or a high-protein (HP) diet (49·5 %) influences metabolism in rainbow trout under challenging intermittent, low dissolved oxygen concentrations. In total, three temperature phases (12, 16, 20°C) were tested sequentially, each of which were split into two oxygen periods with 5 d of unmanipulated oxygen levels (50-70 %), followed by a 5d manipulated oxygen period (16.00-08.00 hours) with low oxygen (40-50 %) levels. For both diets, catabolic protein usage was lowest at 16°C and was not altered under challenging oxygen conditions. Low night-time oxygen elevated mean daily VO2 by 3-14 % compared with the unmanipulated oxygen period for both diets at all temperatures. The relative change in VO2 and retained energy during the intermittent low oxygen period was smaller for the HP diet compared with the SP diet. However, in absolute terms, the SP diet was superior to the HP diet as the former demonstrated 30-40 % lower protein fuel use rates, higher retained energy (1-4 % digestible energy) and slightly lowered VO2 (0-8 %) over the range of conditions tested. The decrease in retained energy under low oxygen conditions suggests that there is scope to improve the performance of SP diets under challenging conditions; however, this study suggests that simply increasing the dietary protein content is not a remedy, and other strategies need to be explored.

Fatty Acid Profile Is Modulated by Dietary Resveratrol in Rainbow Trout (Oncorhynchus mykiss).

To produce fish of a high quality that are rich in omega-3 fatty acids (n-3 FA) and simultaneously generate more sustainable aquaculture, the combined use of phytochemicals and vegetable oils in fish feed seems to be a promising approach. Resveratrol (RV) potentially induces endogenous fatty acid synthesis, resulting in elevated n-3 FA levels in fish. RV putatively influences ∆6-desaturase, the key enzyme in FA metabolism, and serves as a ligand for PPARα, a transcription factor regulating ß-oxidation. Rainbow trout (36.35 ± 0.03 g) were randomly allocated into six groups and fed diets with reduced fish oil levels (F4 = 4%, F2 = 2% and F0 = 0% of dry matter) supplemented with 0.3% (w/w) RV (F4 + RV, F2 + RV and F0 + RV). RV significantly affected FA composition in liver tissue and whole fish homogenates. 20:5n-3 (EPA) and 22:6n-3 (DHA) were significantly increased whereas precursor FA were diminished in fish fed the F2 + RV and F0 + RV diets when compared to F4 + RV and F0. RV significantly elevated ∆6-desaturase protein levels in the livers of F0 + RV fed animals. Hepatic mRNA expression of ∆6-desaturase, PPARα, and its target genes were affected by the dietary fish oil level and not by dietary RV. The results of this study indicated a potential benefit of supplementing RV in fish oil deprived diets elevating n-3 FA levels in rainbow trout.

Bioavailability of free lysine and protein-bound lysine from casein and fishmeal in juvenile turbot (Psetta maxima).

In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (k(Lys)) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25-4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and L-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (P< 0·001). CP, crude lipid and crude ash contents in the whole body were affected by the dietary treatments. The linear regression slope between lysine retention and lysine intake (k(Lys)) was similar between all the dietary lysine sources. The k(Lys) values for the diets supplemented with Cas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7-23·4 mg/kg(0·8) per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

Inland freshwater aquaculture in a warming world.

Climate change potentially threatens the sustainable production of highly valued cold-water fish species in flow-through systems, such as salmonids. By analysing the relationship of water temperature to hydrological characteristics, air temperature, solar exposure, and precipitation, this study predicted temperature dynamics of five temperate cold-water aquaculture facilities under four projected climate change scenarios. Air temperature was found to be directly associated with facility site water temperature, and based on rational assumptions, two of the five facilities were predicted to face critical warming by mid-century. Extreme precipitation events induced acute short-term increases in water temperature of up to 5 °C. Significantly lower warming, roughly equal to the projected climate change-induced increase, was seen with artificial shading lowering temperature by 1 °C. Complementary niche modelling revealed that 37-77 % of current cold-water facilities will likely incur suboptimal climate conditions by the end of the century. Shading of raceways, more efficient water use, and disease management are proposed as key actions to preserve cold-water aquaculture.

Salinity change evokes stress and immune responses in Atlantic salmon with microalgae showing limited potential for dietary mitigation.

Smoltification was found to impact both immune and stress responses of farmed Atlantic salmon (Salmo salar), but little is known about how salinity change affects salmon months after completed smoltification. Here, we examined (1) the effect of salinity change from brackish water to seawater on the stress and immune responses in Atlantic salmon and (2) evaluated if functional diets enriched with microalgae can mitigate stress- and immune-related changes. Groups of Atlantic salmon were fed for 8 weeks with different microalgae-enriched diets in brackish water and were then transferred into seawater. Samples of the head kidney, gill, liver and plasma were taken before seawater transfer (SWT), 20 h after SWT, and 2 weeks after SWT for gene-expression analysis, plasma biochemistry and protein quantification. The salmon showed full osmoregulatory ability upon transfer to seawater reflected by high nkaα1b levels in the gill and tight plasma ion regulation. In the gill, one-third of 44 investigated genes were reduced at either 20 h or 2 weeks in seawater, including genes involved in cytokine signaling (il1b) and antiviral defense (isg15, rsad2, ifit5). In contrast, an acute response after 20 h in SW was apparent in the head kidney reflected by increased plasma stress indicators and induced expression of genes involved in acute-phase response (drtp1), antimicrobial defense (camp) and stress response (hspa5). However, after 2 weeks in seawater, the expression of antiviral genes (isg15, rsad2, znfx1) was reduced in the head kidney. Few genes (camp, clra, c1ql2) in the gill were downregulated by a diet with 8% inclusion of Athrospira platensis. The results of the present study indicate that salinity change months after smoltification evokes molecular stress- and immune responses in Atlantic salmon. However, microalgae-enriched functional diets seem to have only limited potential to mitigate the related changes.

Dietary Chlorella vulgaris supplementation modulates health, microbiota and the response to oxidative stress of Atlantic salmon.

Microalgae are emerging as functional feed ingredients in aquaculture due to their immune-stimulating and stress-modulating properties. We investigated the potential of the microalgae Chlorella vulgaris as a feed supplement to improve the health and modulate microbiota and stress responses of Atlantic salmon. Triplicate groups of Atlantic salmon (~ 126 g) were reared in a recirculating aquaculture system (RAS) at 15 °C and received diets supplemented with 2% (CV2) or 14% (CV14) spray-dried C. vulgaris daily, 14% once weekly (CV14w), or a control diet (CD) for 8 weeks. Subsequently, all groups were exposed to an acute one-hour peracetic acid (CH3CO3H; PAA) treatment, a commonly used disinfectant in RAS. While CV14 increased feed conversion (FCR) significantly, feeding the diets CV2 and CV14w improved protein retention efficiency. CV14 significantly modulated beta-diversity in the intestinal digesta and mucosa, but this effect was already visible in fish fed CV2. Feeding CV14 and, to a lesser degree, CV2 increased the relative abundances of Paenarthrobacter and Trichococcus in the digesta and mucosa, which are able to metabolize complex carbohydrates. However, the same diets reduced the abundance of the lactic acid bacteria Lactobacillus and Weissella in the digesta and Floricoccus in the mucosa. Peracetic acid exposure induced systemic stress (increase in plasma glucose and cortisol) and a local immune response in the gill, with the most prominent upregulation of several immune- and stress-regulated genes (clra, cebpb, marco, tnfrsf14, ikba, c1ql2, drtp1) 18 h after exposure in fish fed the control diet. Fish receiving CV14 once a week showed a reduced transcriptional response to PAA exposure. Catalase protein abundance in the liver increased following exposure to PAA, while superoxide dismutase abundance in the gill and liver was increased in response to C. vulgaris inclusion before stress. Overall, the results highlight that a high (14%) inclusion rate of C. vulgaris in feed for Atlantic salmon impairs feed conversion and shifts the intestinal microbiota composition in digesta and mucosa. Weekly feeding of C. vulgaris proves a viable approach in improving protein retention and improving transcriptional resilience towards oxidative stress in increasingly intensive production systems. Thereby this study may motivate future studies on optimizing temporal feeding schedules for health-promoting aquafeeds.

Anti-PD-(L)1 plus BRAF/MEK inhibitors (triplet therapy) after failure of immune checkpoint inhibition and targeted therapy in patients with advanced melanoma.

BACKGROUND: Effective treatment options are limited for patients with advanced melanoma who have progressed on immune checkpoint inhibitors (ICI) and targeted therapies (TT). Preclinical models support the combination of ICI with TT; however, clinical trials evaluating the efficacy of triplet combinations in first-line setting showed limited advantage compared to TT only. METHODS: We conducted a retrospective, multicenter study, that included patients with advanced melanoma who were treated with BRAF/MEK inhibitors in combination with an anti-PD-(L)1 antibody (triplet therapy) after failure of at least one anti-PD-(L)1-based therapy and one TT in seven major melanoma centers between February 2016 and July 2022. RESULTS: A total of 48 patients were included, of which 32 patients, 66.7% had brain metastases, 37 patients (77.1%) had three or more metastatic organs and 21 patients (43.8%) had three or more treatment lines. The median follow-up time was 31.4 months (IQR, 22.27-40.45 months). The treatment with triplet therapy resulted in an ORR of 35.4% (n = 17) and a DCR of 47.9% (n = 23). The median DOR was 5.9 months (range, 3.39-14.27 months). Patients treated with BRAF/MEK inhibitors as the last treatment line showed a slightly lower ORR (29.6%) compared to patients who received ICI or chemotherapy last (ORR: 42.9%). Grade 3-4 treatment-related adverse events occurred in 25% of patients (n = 12), with seven patients (14.6%) requiring discontinuation of treatment with both or either drug. CONCLUSIONS: Triplet therapy has shown activity in heavily pretreated patients with advanced melanoma and may represent a potential treatment regimen after failure of ICI and TT.

SDA coefficient is temperature dependent in rainbow trout (Oncorhynchus mykiss, Walbaum 1792) in a practical approach using group respirometry.

Rising global temperatures have raised the need for detailed knowledge of the effects of rising temperatures on the physiology of animals used in aquaculture. Here we used a multifactorial bioenergetic approach using groups of rainbow trout (Oncorhynchus mykiss) with an average single fish weight of 183.75 g ± 0.65 g to investigate the interactions of feeding and temperature with key metabolic variables. We used a recirculating aquaculture respirometry system (RARS) to test three ration sizes (0.65; 0.975; 1.3% of live body weight (BW)) over a range of three consecutive temperatures (14; 17; 20 °C). The fish were fed once per day for 6 days at each temperature and subsequently starved for 5 days to return to standard metabolic rate (SMR). This study aimed to answer the highly discussed topic of the temperature dependency of key metabolic specific dynamic action (SDA)-variables SDAcoef and SDAdur. We were able to provide evidence, that in rainbow trout the SDAcoef is highly dependent on the environmental temperature in the first ever approach to assess these variables in a group respirometer with this species. We compared the results of this study with a sophisticated bioenergetic model by Elliot and Hurley (2002) and thereby provide evidence for the practicability of group respirometry as a method to assess bioenergetic data under culture like conditions.

Rapid disease progression on immune checkpoint inhibitors in young patients with stage IV melanoma.

Background: Immune checkpoint inhibitors (ICIs) are the standard of care for metastatic cutaneous melanoma (mCM) patients, but their efficacy in young adults aged less than 40 years remains unclear. Materials and methods: We retrospectively analyzed 303 stage IV melanoma patients of different ages treated with nivolumab, pembrolizumab, or ipilimumab plus nivolumab combination therapy. Clinical data and blood values such as LDH, CRP, and absolute immune cell counts were retrieved from the medical records. Pre-treatment serum concentrations of soluble immune checkpoint proteins were measured using ELISA. In addition, information on frequencies of various T cell subsets in the peripheral blood was collected from a previously reported study (ELEKTRA). Patient characteristics and clinical information was correlated with PFS and OS using univariate and multivariate cox regression analysis. Results: Of 303 patients, 33 (11%) were ≤ 40 years old. The older patients had a median age of 64 (95% CI: 61-66). Concerning prognostic parameters, there was no difference between the age groups, e.g., in gender, LDH, or the existence of brain or liver metastases. Patients aged ≤ 40 years [p = 0.014; HR: 1.6 (95% CI: 1.1-2.4)], presence of liver metastases [p = 0.016; HR: 1.4 (95% CI: 1.0-1.9)], line of ICI treatment [p = 0.009; HR: 1.4 (1.0-1.9)], elevated LDH [p = 0.076; HR: 1.3 (95% CI: 0.97-1.8)], and brain metastasis [p = 0.080; HR: 1.3 (95% CI: 0.97-1.7)], were associated with shorter PFS in univariate analysis. Multivariate analysis revealed that the patient's age (≤ 40 years) remains a high-risk factor upon adjusting for all potential confounders [p = 0.067; HR: 1.5 (95% CI: 0.97-2.3)]. Blood parameters revealed that patients ≤ 40 years have relatively higher frequencies of activated CD4 T cells (CD4 + Ki67 + CD4 + ICOS +) in the blood, and significantly lower number of basophils and CD45RA- memory T cells, compared to patients above 40 years (p < 0.05). In addition, patients ≤ 40 years experiencing disease progression within 6 months of ICI treatment had increased concentrations of sPDL1 (p = 0.05) and sTIM3 (p = 0.054) at baseline. Conclusion: Young patients with stage IV melanoma may experience shorter progression-free survival upon ICI treatment compared to patients above 40 years and are characterized by fewer basophils and memory T cells in the blood.

Dietary carbohydrates induce a higher SDA than lipids in rainbow trout (Oncorhynchus mykiss) based on environmental temperature.

As global temperatures increase so do the needs to investigate how the energy metabolism of fish responds to a broad range of thermal condition. Limited resources make it additionally important to use them sustainably in the feeds for aquaculture. Here we investigated the use of three different carbohydrate to lipid ratios (1:1; 1: 0.6; 1.4: 1 as non-protein energy substrates (NPES) in diets for rainbow trout (Oncorhynchus mykiss) under five different thermal regimes (12; 14; 16; 18; 20 °C) in a bioenergetic approach using a group respirometer. The results showed that the diet with carbohydrate as the main NPES resulted in a quadratic relationship of the specific dynamic action (SDA) values to temperature while diets with lipid as main NPES or a balanced ratio did not show such a response. SDA values in the diet with carbohydrate as the main NPES were significantly higher at temperatures around the optimum (15-17 °C) than the diets with lipid as the main NPES or with a balanced carbohydrate to lipid ratio. The retained energy (RE) was highly dependent on the standard metabolic rate (SMR) and SDA values did not carry over onto them. The protein utilization for energy combustion was significantly lower at 12 °C in the diet with carbohydrate as the main NPES than in the diet with lipid as the main NPES thus indicating that carbohydrates hold a relevant nutritional value especially at lower temperatures.

The microbiota knows: handling-stress and diet transform the microbial landscape in the gut content of rainbow trout in RAS.

BACKGROUND: The aim of the present study was to characterize the effects of handling stress on the microbiota in the intestinal gut contents of rainbow trout (Oncorhynchus mykiss) fed a plant-based diet from two different breeding lines (initial body weights: A: 124.69 g, B: 147.24 g). Diets were formulated in accordance with commercial trout diets differing in their respective protein sources: fishmeal (35% in fishmeal-based diet F, 7% in plant protein-based diet V) and plant-based proteins (47% in diet F, 73% in diet V). Experimental diets were provided for 59 days to all female trout in two separate recirculating aquaculture systems (RASs; mean temperature: A: 15.17 °C ± 0.44, B: 15.42 °C ± 0.38). Half of the fish in each RAS were chased with a fishing net twice per day to induce long-term stress (Group 1), while the other half were not exposed to stress (Group 0). RESULTS: No differences in performance parameters were found between the treatment groups. By using 16S rRNA amplicon sequencing of the hypervariable region V3/V4, we examined the microbial community in the whole intestinal content of fish at the end of the trial. We discovered no significant differences in alpha diversity induced by diet or stress within either genetic trout line. However, the microbial composition was significantly driven by the interaction of stress and diet in trout line A. Otherwise, in trout line B, the main factor was stress. The communities of both breeding lines were predominantly colonized by bacteria from the phyla Fusobacteriota, Firmicutes, Proteobacteria, Actinobacteriota, and Bacteroidota. The most varying and abundant taxa were Firmicutes and Fusobacteriota, whereas at the genus level, Cetobacterium and Mycoplasma were key components in terms of adaptation. In trout line A, Cetobacterium abundance was affected by factor stress, and in trout line B, it was affected by the factor diet. CONCLUSION: We conclude that microbial gut composition, but neither microbial diversity nor fish performance, is highly influenced by stress handling, which also interacts with dietary protein sources. This influence varies between different genetic trout lines and depends on the fish's life history.

Effects of plant-based proteins and handling stress on intestinal mucus microbiota in rainbow trout.

Via 16S rRNA gene amplicon sequencing, this study explores whether the gut mucus microbiota of rainbow trout is affected by the interaction of a plant-protein-based diet and a daily handling stressor (chasing with a fishing net) across two genetic lines (A, B). Initial body weights of fish from lines A and B were 124.7 g and 147.2 g, respectively. Fish were fed 1.5% of body weight per day for 59 days either of two experimental diets, differing in their fish meal [fishmeal-based diet (F): 35%, plant-based diet (V): 7%] and plant-based protein content (diet F: 47%, diet V: 73%). No diet- or stress-related effect on fish performance was observed at the end of the trial. However, we found significantly increased observed ASVs in the intestinal mucus of fish fed diet F compared to diet V. No significant differences in Shannon diversity could be observed between treatments. The autochthonous microbiota in fish fed with diet V was dominated by representatives of the genera Mycoplasma, Cetobacterium, and Ruminococcaceae, whereas Enterobacteriaceae and Photobacterium were significantly associated with diet F. The mucus bacteria in both genetic lines were significantly separated by diet, but neither by stress nor an interaction, as obtained via PERMANOVA. However, pairwise comparisons revealed that the diet effect was only significant in stressed fish. Therefore, our findings indicate that the mucus-associated microbiota is primarily modulated by the protein source, but this modulation is mediated by the stress status of the fish.

Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.

Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

Screening dietary biochanin A, daidzein, equol and genistein for their potential to increase DHA biosynthesis in rainbow trout (Oncorhynchus mykiss).

Plant oil utilization in aquafeeds is still the most practical option, although it decreases the content of the nutritionally highly valuable omega-3 fatty acids eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) in fish. Phytoestrogens and their metabolites are putatively able to affect genes encoding proteins centrally involved in the biosynthesis of EPA and DHA due to their estrogenic potential. Thus, the aim of the study was to screen the potential of the phytoestrogens to stimulate the biosynthesis of EPA and DHA in rainbow trout (Oncorhynchus mykiss). Additionally, the potential effects on growth performance, nutrient composition and hepatic lipid metabolism in rainbow trout were investigated. For that, a vegetable oil based diet served as a control diet (C) and was supplemented with 15 g/kg dry matter of biochanin A (BA), daidzein (DA), genistein (G) and equol (EQ), respectively. These five diets were fed to rainbow trout (initial body weight 83.3 ± 0.4 g) for 52 days. Growth performance and nutrient composition of whole body homogenates were not affected by the dietary treatments. Furthermore, feeding EQ to rainbow trout significantly increased DHA levels by +8% in whole body homogenates compared to samples of fish fed the diet C. A tendency towards increased DHA levels in whole body homogenates was found for fish fed the diet G. Fish fed diets BA and DA lacked these effects. Moreover, EQ and G fed fish showed significantly decreased hepatic mRNA steady state levels for fatty acyl desaturase 2a (delta-6) (fads2a(d6)). In contrast, carnitine palmitoyl transferases 1 (cpt1) hepatic mRNA steady state levels and hepatic Fads2a(d6) protein contents were not affected by the dietary treatment. In conclusion, when combined with dietary vegetable oils, equol and genistein seem to stimulate the biosynthesis of DHA and thereby increase tissue DHA levels in rainbow trout, however, only to a moderate extent.

A diet-change modulates the previously established bacterial gut community in juvenile brown trout (Salmo trutta).

The aim of the present study was to investigate the impact of dietary plant proteins on the gut microbiome of first feeding brown trout (Salmo trutta) reproduced from wild stocks and to evaluate whether the initial microbiome of brown trout fry can be permanently manipulated by the first feeding diet. Therefore, brown trout fry was fed diets based on either 0%, 50% or 90% plant-derived proteins from first feeding onwards and via 16S rRNA gene sequencing a strong dietary influence on the bacterial gut community on phylum and order level was detected. Proteobacteria and Fusobacteria were significantly enhanced when fishmeal was integrated into the experimental diet, whereas plant-derived proteins significantly promoted Firmicutes and Bacteroidetes. In order to evaluate whether the first feeding diet had a permanent effect on the initially established microbial gut community of juvenile brown trout, a cross-over diet-change was applied 61 days post first feeding. 48 days after the diet-change, the gut microbiome of all dietary groups was significantly different from the one initially established after first feeding. Moreover, the first feeding diet had no statistically significant influence on the gut microbiome after the diet-change, demonstrating no permanent effect on the gut microbiome formation.